Project Summary Biochemical Core: Approximately 100 million American adults suffer from chronic pain with a societal cost of approximately $600 billion annually (Institute of Medicine 2011. Opioids effectively alleviate pain for many of these patients, but are limited by the development of adverse events (e.g. nausea, constipation, dependence) and decreased efficacy at tolerable doses over time. Despite a myriad of analgesic compounds on the market, nearly two-thirds of patients report inadequate pain relief while the risks associated with opioid addiction have increased. In the US, prescribed opioids have become the leading abused drugs and top reasons of injury death in the United States. While many studies in the last 15 years concerning opioid abuse have examined the idea that both injury and sustained opioid therapy can induce neuroplastic adaptations, no drug-development strategies have targeted such changes. These changes include endogenous increases in neurokinins, cholecystokinins and enzymes that rapidly degrade endogenous cannabinoids (i.e., FAAH, MAGL) after injury and in some cases after sustained opioid use. Antagonists to the neurokinin receptor-1 and to the CCK receptors-1&2 have been shown to be opioid sparing while decreasing many opioid induced side effects including rewarding behaviors in animals. In addition, many separate studies have identified analgesic efficacy of CB1 and CB2 compounds in animal models of chronic pain, yet no studies have investigated making a bifunctional compound of a CB2 agonist, CB1 neutral antagonist/partial agonist or CB1/CB2 peripherally restricted agonists for chronic pain with reduced unwanted effects. Here we propose to test synthesized compounds with dual activity that includes opioid receptor agonism and either NK-1 (project A), CCK1/2 (project B) antagonism, or CB2-agonist/CB1-antagonist and peripherally restricted mixed CB1/2 agonists (project C) in order to increase analgesic efficacy while inhibiting unwanted side effects with improved drug stability & delivery. The Biochemical Core will provide the necessary screening of novel compounds synthesized by the Chemistry Core based on the knowledge and findings from each of the projects. Due to our years of experience in in vitro and in vivo studies, we will perform 6 aims to efficiently screen all compounds in a highly resourceful manner that will provide feedback to chemistry at all levels in order to develop the best analgesics for chronic pain. These include Aim 1, receptor binding for opioid, NK1, CCK, CB receptors, Aim 2, in vitro functional assays to determine agonist/antagonist activity as well as efficacy, Aim 3, in vivo assays for analgesic/anti-inflammatory activity, Aim 4, in vivo assay in a model of neuropathic pain, Aim 5, in vivo assays for unwanted side effects and Aim 6, In vivo pharmacokinetics. Once compounds have been shown to have efficacy in a model of neuropathic pain (aim 4) the compounds will be tested concurrently in aims 5 and 6. In vitro screening for project C (aims 1 and 2) will be performed in Dr. Makriyannis's laboratory due to having many years of experience in screening of cannabinoid compounds. In vivo screening will be in Biochemical core and labs of Drs. Porreca and Vanderah. These compounds designed to have multiple targets in order to increase analgesic efficacy by addressing endogenous changes due to injury while minimizing the unwanted side effects of opioids alone including opioid addiction. These compounds will entail design for improved plasma stability, CNS penetration, and acceptable pharmacokinetics for movement into clinical trials. The Biochemical Core contains 6 aims in order to properly screen 30-80 novel compounds/yr allowing for a highly efficient structure-activity-relationship (SAR) feedback for improved compound design as noted in our flow chart.